Genetic Diversity and Relationship in Squash Using Morphological, Chemical and Molecular Analyses  

Abd El-Hamed  K. E. , Elwan  M.W. M. , Mohamed  F.H.
Department of Horticulture, Faculty of Agriculture, Suez Canal University, Ismailia, 41522, Egypt
Author    Correspondence author
International Journal of Horticulture, 2015, Vol. 5, No. 12   doi: 10.5376/ijh.2015.05.0012
Received: 18 Jun., 2015    Accepted: 05 Aug., 2015    Published: 25 Oct., 2015
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abd El-Hamed K. E., Elwan M. W.M. and Mohamed F. H., 2015, Genetic Diversity and Relationship in Squash Using Morphological, Chemical and Molecular Analyses International Journal of Horticulture, 2015, Vol.5, No.11 1-10 (doi: 10.5376/ijh.2015.05.0011)


Germplasm characterization is an important link between the conservation and utilization of plant genetic resources. A collection of thirteen summer, spaghetti and acorn squash (Cucurbita pepo L.) and one winter squash (Cucurbita moschata L.) germplasm accessions were screened using morphological (leaf area, fruit weight, fruit length, fruit firmness), chemical [Soluble Solids Content (S.S.C.), ascorbic acid] and molecular [Random Amplified Polymorphic DNA (RAPD)] analyses. A wide range of variability among genotypes was recorded for morphological and chemical characteristics. The percentages of variation were 217%, 532%, 175%, 166%, 148%, 520% for leaf area, fruit length, fruit weight, fruit firmness, S.S.C, ascorbic acid content, respectively. Both significant positive and negative correlations have been found between the morphological and chemical characteristics. The RAPD analysis produced 209 DNA fragments with 100% polymorphism in two or more squash genotypes while none of the fragments showed monomorphic behavior among squash genotypes. At a similarity level of 82% the genotypes were divided into two clusters. The first cluster consisted of eight genotypes (PI 506466- PI 292014- PI 518688- PI 615119- PI 136448- Butternut- Copi- Eskandrani). The second cluster contained only two genotypes (Yellow Crookneck and Shamamy). When the cluster analysis of RAPD patterns was associated with morphological and chemical evaluation of squash genotypes used in this study, there was a notable degree of agreement. The RAPD-PCR was found to be suitable for use with squash due to its ability to discriminate between genotypes and determine their genetic relationship. Results obtained in this investigation provide clear evidence that there is a considerable variation among summer squash genotypes. Present results support the development of breeding programs in squash since high genetic variability in its accessions and landraces has been found.

Cucurbita pepo L.; Cucurbita moschata L.; RAPD; Genotypes characterization; Phylogeny; Clusters.

To increase germplasm usefulness for breeders, morphological and molecular characterization is needed. Morphological markers were useful in distinguishing between ecotypes and identified a high degree of phenotypic variability between populations of several genotypes. The utility of using both molecular and morphological markers has been demonstrated in other species (Duran et al., 2005; Ferguson et al., 2004; Gomez et al., 2004; Tatineni et al., 1996).
In Cucurbita, many landraces cannot be assigned to a given known morphotype; therefore, characterization based on the use of both molecular and morphological markers is essential for elucidating the genetic relationships of ecotypes within this species (Ferriol et al., 2003). In general, the use of both morphological and molecular markers is recommended because each data set provides complementary information with greater power of resolution in genetic diversity analyses (Gomez et al., 2004). The use of both morphological and molecular markers classify genotypes better than employing only one of them when assessing genetic diversity (Franco et al., 2001) and phylogenetic relationships. Both molecular and morphological markers are also valuable for the identification of distinct populations or genotypes for conservation, optimum sites for germplasm collection, and ongoing changes in the pattern of diversity over time. Additionally, morphological and molecular markers are useful for the evaluation and utilization of genetic resources, the study of diversity of pre-breeding and breeding germplasm, and for the protection of the breeder’s intellectual property rights (Franco et al., 2001; Newbury and Ford-Lloyd, 1997).

There are many different molecular techniques available to address diversity-related issues. One popular molecular assay that is based on PCR with arbitrary primers is RAPD (Williams et al., 1990). RAPD is easy and cheap, with no need for DNA probes or sequence information for primer design, very small amounts of DNA (10 ng per reaction) are required, and the procedure is automatable.

Various DNA-sequence polymorphisms have been employed to examine genetic relationships among Cucurbita pepo accessions with high precision. Allozyme polymorphisms revealed a primary division within C. pepo into three subspecies (Decker 1985, 1988). The polymorphism of Cucurbita pepo for fruit shape led to the proposal that this species having eight edible-fruited groups of cultivars (Paris, 1986). Accordingly, these are Acorn, Scallop, Crookneck, Straightneck, Pumpkin, Vegetable Marrow, Cocozelle and Zucchini. The first four cultivar-groups are in C. pepo subsp. texana and the last four are in C. pepo subsp. pepo (Paris et al., 2003).

The germplasm pool of the genus Cucurbita is characterized by abundant diversity (Diez et al., 2002). The enormous morphological diversity of the cultivated races of squash has resulted from variable climate and geographical exposure in which its wild ancestors evolved, coupled with selection pressure (Lira Saade, and Montes Hernandez, 1994). Many sources of exotic and unique germplasm have been discovered and utilized over the years for squash improvement (Paris and Brown, 2005). Traits such as yield, disease resistance, fruit quality have been found and incorporated into current germplasm and have resulted in large improvements in the crop. During the past few decades, enormous effort has been made toward collecting, preserving, and understanding squash germplasm. The most important centers of squash germplasm include National Plant Germplasm System (NPGS), USA and The World Vegetable Center (AVRDC), Taiwan. Genetic stocks consisting of accessions identified as source of resistance to major disease, insect pests, as well as morphological, agronomic characteristics.

Cucurbita is a New World genus of about 20 species; they have been and remain important in diets of world populations ranging from the tropics to warm, temperate regions. Cultivation of the domesticated Cucurbita species has spread beyond their new world origin. Three Cucurbita species, C. pepo, C. moschata, C. maxima comprise the principal cultivated squash and/or pumpkin crops. Both mature and immature fruit are the most important edible plant parts, although for some species; seeds, flowers, roots and even leaves are consumed. Cucurbita pepo L. is believed to be the oldest of the domesticated species. In addition, Cucurbita pepo L. is the most diverse Cucurbita species, has slightly more cold temperature tolerance than other related species (Rubatzky and Yamaguchi, 1997).

Summer Squash (Cucurbita pepo L.) is the most important Cucurbita species, have a mild flavor; eaten raw or cooked and have a short storage life compared to the strongly flavored winter squash. In contrast to summer squash, the postharvest life of winter squashes and pumpkins is much longer and the fruit are not eaten raw. Roasted seeds of some species are a favorite and highly nutritious food.

Fruit quality may include; color, size, firmness, S.S.C., and nutritional value which involve the content of various phytochemicals and vitamins. Ascorbic acid (vitamin C) is one of the most important nutritional quality factors in many horticultural crops and has many biological activities in the human body. More than 90% of the vitamin C in human diets is supplied by fruits and vegetables (Lee and Kader, 2000). The content of vitamin C in fruits and vegetables can be influenced by various factors such as genotypic differences.

The objectives of current investigation were the characterization and analysis of genetic diversity among squash accessions. In addition, the genetic relationship among those accessions and landraces of squash was studied.
Materials and Methods
Plant material
A total of 14 [thirteen summer, spaghetti and acorn squash (Cucurbita pepo L.) and one winter squash (Cucurbita moschata L.)] germplasm collections were analyzed in this study. Eight genotypes were obtained from National Plant Germplasm System (NPGS), USA. Three genotypes were supplied from commercial sources. Three genotypes were obtained from local sources. Names, source, origin and type of all genotypes are presented in Table (1).

 Table 1 Names, source, origin and type of all squash genotypes used in the study

Morphological and chemical evaluation
A Field experiment was conducted at the Experimental Research Farm, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt. The experiment was carried out in spring-summer season of 2014. The soil of the experimental site was sandy soil (85.21% sand, 11.5% silt and 3.29% clay) with pH 8.10 and EC 0.87 dsm-1. Before planting, the experimental location was prepared three months before transplanting. During preparation, a rate of 50 m3 of cattle manure plus 750 kg calcium superphosphate (15.5 % P2O5) per ha were supplemented, then the soil of the site was cleared, ploughed, harrowed and divided into plots. Seeds of squash genotypes were directly sown in soil. Recommended practices for disease and insect control were followed.

Leaf area of different genotypes was recorded using a portable leaf area meter and expressed as (cm2). Fruit weight and fruit length at commercial fresh market maturity of different squash genotypes were manually recorded and expressed as (g) and (cm), respectively. Fruit firmness was measured using a hand Magness Taylor pressure tester and expressed as (Ib/in2) (Mitcham et al., 1996). Soluble Solid Content (S.S.C.) was measured using hand refractometer at 20° and expressed as percent (%) (Mitcham et al., 1996). The extraction and determination of ascorbic acid was performed using the protocol of Pearson (1970) by titration method using 2,6 dichlorophenolindophenol in the presence of oxalic acid and expressed as mg/100 fresh weight (A.O.A.C., 1990).
Statistical analysis:

The experiment was laid-out in a Randomized Complete Block Design (RCBD) with three replications. Data were statistically analyzed using Statistica 6 software (Statsoft, Tulsa, Ok, USA) with mean values compared using Duncanś multiple range with a significance level of at least p ≤ 0.05.

Genomic DNA isolation, PCR reaction and RAPD analysis
DNA of 14 squash genotypes of different geographical origin was extracted from the recent leaves of the plants as described by Murray and Thompson (1980). Ten random oligonucleotide (10 mer) primers were tested for use in RAPD analysis. The primers were (A01, A02, A03, A04, A05, A06, A07, A08, A09, and A10) (Laboratories of the Midland Certified Reagent Company Inc. Texas, USA) (Table 2).

Table 2 The nucleotide sequence of primers used in the RAPD analysis 

The PCR reaction were carried out in 50 μL volumes tubes containing 100 ng of genomic DNA, 10 μM of each primer, 200 μM of dATP, dDTP, dCTP, dGTP, 10 mM Tris-HCL, pH 8.3, 50mM MgCl2 and 0.001% gelatin. The Taq DNA polymerase (Promega, Corporation, Madison, WI) concentration was 1.5 units per assay. The PCR reaction was conducted using Eppendorf thermocycler programmed according to the following protocol that consisted of 1 min. at 95 Cº followed by 55 cycles of 20 sec. at 94ºC, 30 sec. at 37 ºC, and 2 min. at 72 ºC as described by Nadig et al., (1998). Amplification products were electrophoresed in 1.5% Agarose gel in 1 x TAE buffer, stained with ethidium bromide and visualized with UV transilluminator and photographed- A 100 bp DNA ladder of 1000 bp (Promega, Corporation, Madison, WI) was used as a standard for primers (Gene on, UK).

Analytical procedures
Fragments that were clearly resolved on the gels were scored as 1 or 0 (i.e., present or absent, respectively) across all the fourteen genotypes. Bands that could not be confidently scored were regarded as missing data. The data matrices of Euclidean distances were analyzed by the STATSOFT ver. 6 (2001) and similarities between genotypes were estimated using the simple matching coefficient (Sokal and Michener, 1958), calculated as Sij = a+d/n where (a) is the number of positive matches (i.e. the presence of a band in both samples), (d) is the number of negative matches (i.e. the absence of a band in both samples), (n) is the total number of bands. The program generated a dendrogram, which grouped the genotypes using unweighted pair group method with arithmetic average (UPGMA) with the JOINING tree clustering module.

Morphological and chemical characteristics
The entire studied genotypes exhibited a high variability for all the morphological and chemical characteristics. A wide range of values among genotypes was recorded in all traits.

Concerning leaf area, the percentage of variation (maximum/minimum*100) was high with 217%. Genotypes with Egyptian source were characterized by high leaf area. Genotype (Copi) was the highest genotype with 425 cm2 more than twice the lowest genotype (PI 518687) with 196.11 cm2. Also, other Egyptian genotypes (Shmamay and Eskandrani) ranked high for leaf area (Table 3). Regarding fruit weight, the percentage of variation was extremely high with 532%. The winter squash genotype (Butternut) ranked as the first genotype for this trait with 283.44 g. Accessions (PI 518687 and PI 518688) which belong to acorn squash came after Butternut but was significantly different with 153 and 147.56 g respectively (Table 3). The group of genotypes with which belong to acorn squash came after Butternut but was significantly different with 153 and 147.56 g respectively (Table 3). The group of genotypes with Egyptian origin maintain high fruit weight compared with other summer squash genotypes. The percentage of variation for fruit length was relatively moderate with 175%. The acorn squash genotypes (PI 518687 and PI 518688) had the higher fruit length followed by the winter squash genotype (Butternut) and the commercial genotype (Eskandrani) with none significant difference (Table 3). The percentage of variation for fruit firmness was moderate with 166%. Again, acorn squash genotype (PI 518687) ranked with the highest fruit firmness among all tested genotypes followed by winter squash (Butternut) and genotypes with Egyptian source (Eskandrani and Copi) (Table 3). The range for S.S.C among tested genotypes was relatively limited with percentage of variation as 148%. Acorn squash genotype (PI 518688) was the highest genotype concerning S.S.C with 5.07% but was not significantly different than the following genotype (Butternut) with 4.98% (Table 3). The range for ascorbic acid content among tested genotypes was broad with percentage of variation as 520%. There was no clear trend concerning ascorbic acid content among tested genotypes and none of the morphotypes showed apparent association with ascorbic acid content (Table 3). 

Table 3 Morphological (leaf area, fruit weight, fruit length, fruit firmness), chemical (S.S.C., ascorbic acid) characterization of the fourteen squash genotypes evaluated in this study 

The results in Table (4) and Figure (1) revealed a significant negative correlations between fruit length and fruit weight (r = -0.39) as well as fruit firmness and S.S.C. content (r = -0.2). However, a significant positive correlation has been found between leaf area and both fruit length (r = 0.46) and ascorbic acid content (r = 0.42). In addition, a significant positive correlation has been also found between fruit length and ascorbic acid content (r = 0.29). Non-significant correlation coefficients were observed among other morphological and chemical traits (Table 4).

Table 4 Phenotypic correlation (r) and significance level between morphological and chemical characters in 14 squash genotype 


Figure 1 Correlation between leaf area and fruit length (a), fruit weight and fruit length (b), leaf area and ascorbic acid content (c), fruit length and ascorbic acid content (d), fruit firmness and S.S.C. (e) 

Molecular evaluation
This study was carried out to apply molecular tools to assess the polymorphism existed as well as determine the genetic relationship among 14 squash genotypes. Ten primers which produced good and reproducible polymorphic bands among the 14 squash genotypes were used for further analysis. These 10 primers produced 209 DNA fragments with 100% polymorphism in two or more squash genotypes while none of the fragments showed monomorphic behavior among squash genotypes (Figure 2) (Table 5).The highest number of bands with polymorphism was observed with primer A01 (with 45 bands), while, the lowest primer was A10 (with 3 bands). The similarity matrix showed that the lowest similarity (0.674) was between the genotypes PI 506467 and PI 518687, while the highest similarity (0.891) was between the genotypes Copi and Eskandrani and also between genotypes PI 518688 and PI 615119 (Table 6). According to the dendrogram (Figure 3), at a similarity level of 82% the genotypes were divided into two clusters. The first cluster consisted of eight genotypes (PI 506466- PI 292014- PI 518688- PI 615119- PI 136448- Butternut- Copi- Eskandrani). The second cluster contained only two genotypes Yellow Crookneck and Shamamy

Table 5 List of the primers and the degree of the polymorphism obtained among the 14 squash genotypes 


Table 6 Similarity coefficient matrix among the 14 squash genotypes based on 10 RAPDs markers 


Figure 2 The RAPD markers generated by primers number 1,4,7, and 9 on the tested genotypes 

When the cluster analysis of RAPD patterns was associated with morphological and chemical evaluation of squash genotypes used in this study, there was a notable degree of agreement (Table 1 and 3) (Figure 3). From the dendrogram, genotypes Copi and Eskandrani showed a high degree of similarity according to RAPD data and both belong to the same Egyptian origin. In addition, both genotypes possess non-significantly different morphological and chemical characters such as plant leaf area, fruit firmness and fruit S.S.C. and similar fruit length and weight.

Figure 3 UPGMA dendrogram of the 14 squash genotypes based on the 10 random RAPD primers. G1: PI 506466; G2: PI 292014 

Molecular techniques have revolutionized the ability to characterize genetic materials and helped provide knowledge of genetic diversity and means of making predictions of how diversity may change (Karp et al., 1997). Molecular tools have significant applications in classification, identification and evaluating genetic resources as well as understanding the structure and history of diversity (Karp, 2000).

Knowledge of the genetic diversity of a crop is essential for the parental selection in order to maximize genetic improvement. More accurate and complete descriptions of the genotypes and patterns of the genetic diversity could help determine future breeding strategies and facilitate the introgression of diverse germplasm into the current commercial summer squash genetic base. Genetic variation in traditional landraces is the crucial genetic pool for plant breeding. Evaluation of the landraces in terms of phenotypic behavior and genetic variability needs to be performed before they are used in breeding programs to develop new cultivars that are more productive and of greater nutritional value. The identification and use of landraces of diverse genetic background is a critical issue for successful breeding programs through the selection of suitable parents. Many studies on molecular level have been carried out using different molecular markers such as, RAPD (Tsivelikas et al., 2009), AFLP (Ferriol et al., 2003), SSR (Barzegar et al., 2013), ISSR and SRAP (Inan et al., 2012) to study genetic diversity within and among several species of Cucurbita. The majority of previous studies used commercial cultivars. This study provide detailed morphological, chemical and molecular characterization of a germplasm collection of Cucurbita pepo L. made up of diverse set of plant introduction accessions, commercial cultivars and landraces, which is expected to increase the genetic basis of squash. There is no information about the genetic diversity among Cucurbita landraces that are cultivated in Egypt and they are poorly characterized so far.

Results obtained in our investigation provide clear evidence that there is a considerable variation among summer squash genotypes. Present results support the development of breeding programs in Cucurbita pepo since we have found high genetic variability in its accessions and landraces.

Plant leaf area is affecting light absorption and consequently photosynthesis. The amount and intensity of light during the growing season have a definite influence on the amount of ascorbic acid formed (Lee and Kader, 2000). Ascorbic acid is synthesized from sugars supplied through photosynthesis in plants which can explain the positive correlation between leaf area and ascorbic acid content (r = 0.42). The significant negative correlation between fruit firmness and S.S.C. content (r = -0.2) observed in this investigation concerning squash was also found in other crops. A highly significant negative correlation has been detected between fruit firmness and S.S.C. in apple (Kvikliene et al., 2006). However, different results were recorded in peach and nectarine (Cantín et al., 2010). The lack of correlation between traits in such studies should have been no surprise since these genotypes differ genetically in many aspects. The non-significant correlation between fruit weight and S.S.C. for squash in this study was also observed in pepper (do Rêgo et al., 2011) and in peach and nectarine (Cantín et al., 2010).The high leaf area revealed in Egyptian genotypes is in agreement with known knowledge concerning the high vegetative growth of such local genotypes. The heavy weight of fruits of winter and acorn squash compared with summer squash are in accordance with established information in Cucurbita spp. (Rubatzky and Yamaguchi 1997). The high fruit weight of Egyptian genotypes is due to the selection pressure performed on such genotypes for superior fruits for long time (El-Hadi et al., 2014). The high fruit firmness for acorn and winter squash is due to the hard nature of fruits in such morphotypes (Rubatzky and Yamaguchi 1997). Although, the range for ascorbic acid content among tested genotypes was wide with percentage of variation as 520%, there was no clear trend among tested squash morphotypes. This may be explained because of the overall low level of ascorbic acid content in squash compared with other vegetables such as pepper, broccoli, strawberry and spinach (Lee and Kader 2000).

Our results suggest that RAPD markers are good alternative for the evaluation of genetic diversity and assessing the separation among landraces belonging to different geographical regions. The level of polymorphism among the Cucurbita pepo L. genotypes was extremely high. This high level of RAPD markers polymorphism in Cucurbita pepo genotypes is in accordance with the results of Hadia et al. (2008) and Tsivelikas et al. (2009), who reported that Cucurbita pepo is a highly polymorphic species. In an earlier study, Hadia et al. (2008) reported 89% polymorphism among Cucurbita pepo genotypes. Lower percentage of polymorphism (61%) has been detected in Cucurbita pepo by El-Adl et al., (2012) which can be due to the low number of primers (6 primers) and genotypes (7 genotypes) used in such study. Also, a low level of polymorphism in Cucurbita pepo was described by Al-Tamimi, (2014) and most likely for using low number of primers and genotypes.

The variation assessed by 209 polymorphic RAPD bands generated by 10 primers that screened the 14 genotypes. The DNA fragments generated by RAPD primers were different in number, intensity and position indicating high genetic variation among the studied genotypes. Primer A01 provided the highest number of polymorphic bands (45); whereas, primer A10 provided the lowest number of polymorphic bands (3). The variation among reproducible bands generated by each primer depends on the primer sequence and the extent of variation in the specific genotypes (Upadhyay et al., 2004).

It is apparent from our results that genotypes Copi and Eskandrani shared the highest number of RAPD markers using the 10 primers used in this study which represented also in the high similarity matrix coefficient (0.891). This piece of information suggests that their parental breeding lines were genetically close to each other. Genotype Copi is maintained by local farmers and it can be suggested that it is originated as segregation population out of genotype Eskandrani.

The high correlation between the similarity matrix and the UPGMA dendrogram, indicates a good representation of the relationship among the genotypes. On the basis of the comparison between morphological and chemical evaluation and RAPD molecular markers results, a great deal of correspondence has been found. The obtained clustering based on RAPD was consistent with morphological and chemical characteristics of the squash genotypes used in this study. However, in few cases the clusters were not in agreement with the known genetic background of such genotypes.

For instance, genotype Butternut which belong to the winter squash (Cucurbita moschata L.) was assigned to a cluster with other summer squash genotypes which belong to (Cucurbita pepo L.) while, few summer squash genotypes fall outside that cluster and appear as distantly related to the rest of (Cucurbita pepo L.) genotypes.

The RAPD assay has been successfully used in taxonomic and genetic diversity studies of squash (Al-Tamimi, 2014; El-Adl et al., 2012; Hadia et al., 2008; Tsivelikas et al., 2009; Ntuli et al., 2013; Baranek et al., 2000; Radwan, 2014). The technique was found by former and by us to be suitable for use with squash due to its ability to generate reproducible polymorphic markers. Our study reports an additional 10 RAPD markers capable of distinguishing among the squash genotypes, and extends the application of those markers to the identification of the genetic relationships as well as diversity and structure present within those genotypes.

Germplasm characterization is an essential link between the conservation and utilization of plant genetic resources. Molecular DNA techniques allow researchers to identify accessions at the taxonomic level, assess the relative diversity within and among species and locate diverse accessions for breeding purposes. Plant breeders usually use efficient molecular marker tools such as RAPD-PCR to organize their genetic resources into related groups to make more informed decisions regarding choice of parents in breeding programs. Knowledge of genetic relationships when complemented with phenotypic data can reveal source of desirable characteristics in more closely related genotypes which permit the recovery of the phenotype of the recurrent parents in fewer breeding generations than would be required for a more distantly related donor parent. Knowledge of wide genetic diversity observed in squash provides information that is important in the management of germplasm resources for future breeding programmes.

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International Journal of Horticulture
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